It has long been thought that suppressor cells play a role in the progression of cancer (Dye et al., J. Exp. Med. 154:1033-1042 (1981)). In fact, active suppression by T regulatory cells plays an important role in the down-regulation of T cell responses to foreign and self-antigens.
T cells are a class of lymphocytes, having specific T cell receptors (TCRs) that are produced as a result of gene rearrangement. T cells have diverse roles, which are accomplished by the differentiation of distinct subsets of T cells, recognizable by discrete patterns of gene expression. Several major T cell subsets are recognized based on receptor expression, such as TCR-[alpha]/[beta], and TCR [gamma]/[delta] and invariant natural killer cells. Other T cell subsets are defined by the surface molecules and cytokines secreted therefrom.
For example, T helper cells (CD4 cells) secrete cytokines, and help B cells and cytotoxic T cells to survive and carry out effector functions. Cytotoxic T cells (CTLs) are generally CD8 cells, and they are specialized to kill target cells, such as infected cells or tumor cells. Natural killer (NK) cells are related to T cells, but do not have TCRs, and have a shorter lifespan, although they do share some functions with T cells and are able to secrete cytokines and kill some kinds of target cells.
Human and mouse peripheral blood contains a small population of T cell lymphocytes that express the T regulatory phenotype (“Treg”), i.e., positive for both CD4 and CD25 antigens (i.e., those CD4-positive T cells that are also distinctly positive for CD25). First characterized in mice, where they constitute 6-10% of lymph node and splenic CD4-positive T cell populations, this population of CD4-positive CD25-positive cells represents approximately only 5-10% of human peripheral blood mononuclear cells (PBMC), or 2-7% of CD4-positive T cells, although some donors exhibit a more distinct population of CD4-positive and CD25-positive cells. About 1-2% of human peripheral blood PBMCs are both CD4 positive (CD4-positive) and CD25 brightly positive (CD25-positive) cells.
There are several subsets of Treg cells (Bluestone et al., Nature Rev. Immunol. 3:253 (2003)). One subset of regulatory cells develops in the thymus. Thymic derived Treg cells function by a cytokine-independent mechanism, which involves cell to cell contact (Shevach, Nature Rev. Immunol 2:389 (2002)). They are essential for the induction and maintenance of self-tolerance and for the prevention of autoimmunity (Shevach, Annu. Rev. Immunol. 18:423-449 (2000); Stephens et al., 2001; Turns et al., 2001; Thornton et al., 1998; Salomon et al., Immunity 12:431-440 (2000); Sakaguchi et al., Immunol. Rev. 182:18-32 (2001)).
These professional regulatory cells prevent the activation and proliferation of autoreactive T cells that have escaped thymic deletion or recognize extrathymic antigens, thus they are critical for homeostasis and immune regulation, as well as for protecting the host against the development of autoimmunity (Suri-Payer et al., J. Immunol. 157:1799-1805 (1996); Asano et al., J. Exp. Med. 184:387-396 (1996); Bonomo et al., J. Immunol. 154:6602-6611 (1995); Willerford et al., Immunity 3:521-530 (1995); Takahashi et al., Int. Immunol. 10:1969-1980 (1998); Salomon et al., Immunity 12:431-440 (2000); Read et al., J. Exp. Med. 192:295-302 (2000). Thus, immune regulatory CD4-positive CD25-positive T cells are often referred to as “professional suppressor cells.”
However, Treg cells can also be generated by the activation of mature, peripheral CD4-positive T cells. Studies have indicated that peripherally derived Treg cells mediate their inhibitory activities by producing immunosuppressive cytokines, such as transforming growth factor-beta (TGF-[beta]) and IL-10 (Kingsley et al., J. Immunol. 168:1080 (2002); Nakamura et al., J. Exp. Med. 194:629-644 (2001)). After antigen-specific activation, these Treg cells can non-specifically suppress proliferation of either CD4-positive or CD25-positive T cells (demonstrated by FACS sorting in low dose immobilized anti-CD3 mAb-based co-culture suppressor assays by Baecher-Allan et al., J. Immunol. 167(3):1245-1253 (2001)).
Studies have shown that CD4-positive CD25-positive cells are able to inhibit anti-CD3 stimulation of T cells when co-cultured with autologous antigen presenting cells (APC), but only through direct contact (Stephens et al., Eur. J. Immunol. 31:1247-1254 (2001); Taams et al., Eur. J. Immunol. 31:1122-1131 (2001); Thornton et al., J. Exp. Med. 188:287-296 (1998)). However, in mice this inhibitory effect was not able to overcome direct T cell stimulation with immobilized anti-CD3 or with anti-CD3/CD28 (Thornton et al., 1998). In previous reports, human CD4-positive CD25-positive T cells isolated from peripheral blood required pre-activation in order to reveal their suppressive properties, as direct culture of the regulatory cells was generally insufficient to mediate suppressive effects (Dieckmann et al., J. Exp. Med. 193:1303-1310 (2001)).
Others have also found that the inhibitory properties of human CD4-positive CD25-positive T cells are activation-dependent, but antigen-nonspecific (Jonuleit et al., J. Exp. Med. 193:1285-1294 (2001); Levings et al., J. Exp. Med. 193(11):1295-1302 (2001); Yamagiwa et al., J. Immunol. 166:7282-7289 (2001)), and have demonstrated constitutive expression of intracellular stores of cytotoxic T lymphocyte antigen-4 (CTLA-4) (Jonuleit et al., 2001; Read et al., J. Exp. Med. 192:295-302 (2000); Yamagiwa et al., 2001; Takahashi et al., J. Exp. Med. 192:303-310 (2000)). Moreover, after T-cell receptor (TCR)-mediated stimulation, CD4-positive CD25-positive T cells suppress the activation of naive CD4-positive CD25-negative T cells activated by alloantigens and mitogens (Jonuleit et al., 2001).
Both mouse and human Treg cells express CTLA-4, however the role of CTLA-4 in tolerance induction and its capacity to impart inhibitory function to regulatory CD4-positive CD25-positive T cells is controversial. CTLA-4 (also known as CD152) is a homolog of CD28 and is a receptor for the CD80 and CD86 ligands. CTLA-4 inhibits T cell responses in an antigen and TCR-dependent manner. T cells that have impaired CTLA-4 function have enhanced T cell proliferation and cytokine production. In contrast, enhanced CTLA-4 function leads to inhibited cytokine secretion and impaired cell cycle progression both in vitro and in vivo. In the mouse, CTLA-4 is not required for suppressive function of the Treg cells, as opposed to its requirement in humans.
A recent study has shown that Treg cells grow extensively in vivo (Tang, J. Immunol. 171:3348 (2003)), while others have suggested that the efficacy of therapeutic cancer vaccination in mice can be enhanced by removing CD4-positive CD25-positive T cells (Sutmuller et al., J. Exp. Med. 194:823-832 (2001)). Studies have also indicated that depletion of regulatory cells led to increased tumor-specific immune responses and eradication of tumors in otherwise non-responding animals (Onizuka et al., Cancer Res. 59:3128-3133 (1999); Shimizu et al., J. Immunol. 163:5211-5218 (1999)). Susceptible mouse strains that were made CD4-positive CD25-positive deficient by neonatal thymectomy were shown to develop a wide spectrum of organ-specific autoimmunities that could be prevented by an infusion of CD4-positive CD25-positive T cells by 10-14 days of age (Suri-Payer et al., J. Immunol. 160:1212-1218 (1998)). That study also found that CD4-positive CD25-positive T cells could inhibit autoimmunity induced by autoantigen-specific T cell clones. The transfer of CD4-positive CD25-negative T cells into nude mice also reportedly led to the development of autoimmune disorders which could be prevented by the co-transfer of CD4-positive CD25-positive T cells using lymphocytes first depleted of CD25-positive cells (Sakaguchi et al., J. Immunol. 155:1151-1164 (1995)).
Hereafter, the transcription factor Forkhead box P3 (FoxP3) was related to the generation and fuction of naturally occurring Treg. Mice in which FoxP3 protein was deleted due to a mutation in the FoxP3 gene, developed severe autoimmune syndroms and wasting diseases (socalled “scurfy” mice; Brunkow et al., Nat Genet. 27:68-73, 2001). This seminal discovery enabled to attribute the cause of the X-linked IPEX syndrome (Immunodysregulation, Polyendocrinopathy, and Enteropathy, X-linked) in humans to a mutation in the FoxP3 gene (Bennett et al. Nat Genet. 27: 20-21; 2001). Later studies also demonstrated the presence of FoxP3 in some adaptive Treg subsets.
However, data also indicate that the role of CD4-positive CD25-positive cells is not limited to self-tolerance and the prevention of autoimmunity. While few studies have addressed the role of CD4-positive CD25-positive T cells in alloresponses or in transplantation, CD4-positive CD25-positive T cells have been reported to prevent allograft rejection, both in vitro and in vivo (Hara et al., J. Immunol. 166:3789-3796 (2001); Taylor et al., J. Exp. Med. 193:1311-1318 (2001)). Allogeneic stimulation of human T cell proliferation is also blocked by CD4-positive CD25-positive T cells (Yamagiwa et al., 2001), whereas Wood's laboratory has shown that CD4-positive CD25-positive T cells suppress mixed lymphocyte responses (MLR), but only when the alloantigen was presented by the indirect, and not the direct, pathway of allorecognition (Hara et al., 2001). It is likely that direct antigen presentation occurs between the regulatory T cells and the anti-CD3/28 stimulated responder T cells, as the sorted CD4-positive 25-positive cells are highly depleted of professional APC.
The absence of Tregs or depletion of Tregs is shown to result in the development of auto-immunity, such as Type 1 Diabetes, Inflammatory bowel disease (IBD), thyroididites, Multiple Sclerosis and Systemic lupus erythematosus (SLE). Moreover the disease can be reversed by the adoptive transfer of CD4+CD25+Treg cells. Besides a deficiency in Treg number, T cell regulation in autoimmunity has also been shown to fail due to a deficiency in the function of Treg to inhibit effector T cells. It is clear that defects in Treg cell number and function can contribute to disease and therapies directed at these defects have the potential to prevent and also cure these diseases. Animal studies suggest that an increase in Treg cell number at the site of inflammation is likely to be therapeutic in autoimmunity. This can be achieved by adoptive transfer of in-vitro expanded autologous Tregs or by the use of agents that promote Treg cell proliferation, survival and induction. The identity of factors that influence cell number and function of Tregs are not clearly identified at the moment, and may be crucial for the application of autoimmune diseases.
Antigen Presenting cells such as DC are known for their capacity to differentiate naive CD4 T cells into different lineage of T cells, such as Th1, Th2, Th17 and Treg. Recent studies demonstrate that a population of gut DC, particularly lamina propria CD103+ DCs, can promote the conversion of naive CD4+ T cells into FoxP3+ iTregs through the secretion of retinoic acid (RA) in conjunction with TGF-β. DC express various receptors such as CD80/86 that can be bound by CTLA-4 on Tregs that triggers the induction of the enzyme indoleamine 2,3 dioxygenase (IDO) in DC. IDO converts tryptophan into pro-apoptotic metabolites that suppress effector T cells. On the other hand engagement of MHC class II on DC by LAG3 on Tregs suppresses APC maturation and reduces their ability to activate T cells. These findings demonstrate that DC may differentiate CD4 T cells into Tregs. However, little is still known on the mechanism and signals that reach DC to instruct CD4 naïve T cells to differentiate into Tregs.
Patients suffering from autoimmune diseases or inflammatory diseases would greatly benefit from treatments wherein the Treg numbers or function are improved.
Applicants have established that the uptake of specific glycosylated antigens by DCs regulates the number and function of Tregs. This opens new opportunities for the treatment of unwanted immune reactions and leads to new methods and means for the treatment of autoimmune diseases and inflammatory diseases.